Von Willebrand factor is a multimeric protein in plasma that plays an essential role in primary hemostasis by mediating platelet adhesion at sites of vascular injury. The hemostatic function of von Willebrand factor depends on the degree of multimerization of its subunits. A fraction of the von Willebrand factor secreted from endothelial cells and platelets contains a highly polymerized form of von Willebrand factor that would spontaneously agglutinate platelets to form pathological occlusive thrombi in the circulation. These highly polymerized forms of von Willebrand factor are proteolytically processed to a less polymerized form in a shear-dependent manner by the metalloprotease ADAMTS13 in plasma. Thus, post-secretion processing of von Willebrand factor in plasma is a critical step in regulating the function of von Willebrand factor. Although shear stress and the presence of ADAMTS13 are essential for processing, how this process is regulated is not known. Studies proposed in this application will focus on how intramolecular interactions among the various domains within the von Willebrand factor subunit, and intermolecular interactions between von Willebrand factor and other macromolecules, including that of a cofactor, regulate the proteolytic processing of von Willebrand factor by ADAMTS13. In aim 1, by use of recombinant and mutant fragments of von Willebrand factor, we will determine the role of the ristocetin binding site in regulating the accessibility of the A2 domain for cleavage by ADAMTS13. In aim 2, we will study how allosteric interactions among the three 'A'domains within the von Willebrand factor subunit and their interactions with other macromolecules modulate the exposure and accessibility of the cleavage site. In aim 3, we will characterize how heparin and a heparin-like cofactor regulate the cleavage of von Willebrand factor by ADAMTS13. An improved understanding of this process would lead to new strategies for the prevention and treatment of thrombotic and bleeding diseases caused by aberrant, inadequate or excessive processing of von Willebrand factor. PUBLIC HEALTH RELEVANCE: The proposed research is focused on understanding how a plasma metalloprotease regulates the function of the adhesive protein von Willebrand factor, which initiates platelet adhesion and aggregation at the site of vascular injury. These studies will further our understanding of how the metalloprotease and a newly identified cofactor regulate the function of von Willebrand factor and thrombi formation in health and in disease.